Fig. 7. Binding of 14-3-3s to HA-PFK-2 in myristylated PKB-transfected HeLa cells. HeLa cells were transfected with plasmid pCMV5-PFK-2 to express HA-PFK-2 (A), or pGFPPFK2 to express GFP–HA-PFK-2 (B). Where indicated, cells were co-transfected with a plasmid expressing PKB with a myristylation consensus site (pCMV5-PKBmyr). (A) Cell extracts (30 µg) were analysed for binding to DIG-14-3-3s (14-3-3 overlay), phospho-specific antibodies that recognize pSer483 and pSer466 on cardiac PFK-2 and anti-HA antibodies, and phospho-specific antibodies against pSer473 on PKBα and anti-PKB. (B) GFP–HA-PFK-2 and associated proteins were precipitated from cell extracts (500 µg) with 3 µg anti-GFP antibodies (20 µl of protein G–Sepharose). Washed anti-GFP immunoprecipitates were subjected to SDS–PAGE, transferred to nitrocellulose and blotted with K19 antibodies that recognize all seven human 14-3-3 isoforms and anti-GFP antibodies.